Developing a Photoautotrophic Micropropagation System for Woody Plants

in vitro including cotyledonary stage somatic embryos have the ability to grow photoautotrophically (without sugar in the culture medium), and that the low or negative net photosynthetic rate of plants in vitro is due not to poor photosynthetic ability, but to the low CO₂ concentration in the air-tight culture vessel during the photoperiod. Furthermore, we have shown that the photoautotrophic growth of several woody plants in vitro can be significantly promoted by increasing the CO₂ concentration and light intensity in the vessel, by decreasing the relative humidity in the vessel, and by using a fibrous or porous supporting material with high air porosity instead of gelling agents such as agar. In this paper, the advantages of photoautotrophic micropropagation in a conventional, small culture vessel with a microporous gas filter for enhancing natural ventilation and in a large culture vessel with a forced ventilation unit are described for woody plants such as acacia (Acacia mangium), coffee (Coffea arabusta), eucalyptus (Eucalyptus camaldlensis), mangosteen (Garcinia mangostana), neem (Azadirachta indica), paulownia (Paulownia fortunei), and pine (Pinus radiata). Received 30 August 2001/ Accepted in revised form 27 September 2001     

Fringe mode transmittance laser Doppler microscope anemometer: its adaptation for measurement in the microcirculation

Blood flow analysis in the microcirculation requires accurate measurement of velocity, volume flow and shear-rate versus shear-stress relationships. The resolution of most anemometers is too limited to obtain useful measurements, especially near the blood vessel wall and at branches and bifurcations. To make such measurements possible with a noninvasive, high resolution, accurate technique, we have developed a fringe mode, transmittance laser Doppler microscope anemometer (LDMA). This system has an intrinsically high spatial resolution (10 × 12 μm), and does not require a high concentration (10⁶/cm³) of scatterers or red blood cells (RBC) as in our application. Preliminary measurements of water flow in a rectangular channel were conducted to ascertain the reliability and accuracy of velocity measurements using the LDMA. Velocity profiles were then measured by the LDMA system in arterioles 38–135 μm in diameter, in the transparent, everted cheek pouch of the anaesthelized hamster. The extremely high resolution of the optical system, and the ultra-fine traversing mechanism of the microscope slage, made velocity readings larger than 0.02 mm/s with accuracy and reproducibility better than 1%, possible near the wall to within 7–10 μm.     

Functional lifespans of xylem vessels: Development,hydraulic function,and post‐function of vessels in several species of woody plants

Premise of the Study

Xylem vessels transition through different stages during their functional lifespan, including expansion and development of vessel elements, transition to vessel hydraulic functionality, and eventual transition to post‐functionality. We used information on vessel development and function to develop a model of vessel lifespan for woody plants.

Methods

We examined vessel functional lifespan using repeated anatomical sampling throughout the growing season, combined with active‐xylem staining to evaluate vessel hydraulic transport functionality. These data were combined with a literature review. The transitions between vessel functional lifespans for several species are illustrated, including grapevine (Vitis vinifera L., Vitaceae), English oak (Quercus robur L., Fagaceae), American chestnut [Castanea dentata (Marshall) Borkh.; Fagaceae], and several arid and semi‐arid shrub species.

Key Results

In intact woody plants, development and maturation of vessel elements may be gradual. Once hydraulically functional, vessel elements connect to form a vessel network that is responsible for bulk hydraulic flow through the xylem. Vessels become nonfunctional due to the formation of gas emboli. In some species and under some conditions, vessel functionality of embolized conduits may be restored through refilling. Blockages, such as tyloses, gels, or gums, indicate permanent losses in hydraulic functional capacity; however, there may be some interesting exceptions to permanent loss of functionality for gel‐based blockages.

Conclusions

The gradual development and maturation of vessel elements in woody plants, variation in the onset of functionality between different populations of vessels throughout the growing season, and differences in the timing of vessel transitions to post‐functionality are important aspects of plant hydraulic function.     

High photosynthetic photon flux and high CO2 concentration under increased number of air exchanges promote growth and photosynthesis of four kinds of orchid plantlets in vitro

Summary In vitro plantlets of Phalaenopsis ‘Happy Valentine’, Neofinetia falcate Hu, Cymbidium kanran Makino, and Cymbidium goeringii Reichb. f. were grown under photoautotrophic [high photosynthetic photon flux (PPF), high CO₂ concentration, and increased number of air exchanges] and heterotrophic (low PPF, low CO₂ concentration, no air exchanges) culture conditions. After 40 d of culture, a significant difference in plantlet growth was observed between the two cultures. Total fresh and dry mass were on average 1.5 times greater in photoautotrophic culture than in heterotrophic culture. Higher net photosynthetic rates were also observed for Phalaenopsis in photoautotrophic culture. In photoautotrophic culture, little difference was observed in air temperature between the inside and outside of the culture vessel, whereas in heterotrophic culture, air temperature inside the culture vessel was 1–2°C higher than that outside the culture vessel. Relative humidity inside the culture vessel was remarkably different between the two cultures: 83–85% in photoautotrophic culture and 97–99% in heterotrophic culture. These results indicated that growth and net photosynthetic rate of in vitro orchid plantlets were susceptible to the culture environments such as PPF, CO₂ concentration, relative humidity (RH), and the number of air exchanges, which would allow a more efficient micropropagation system for these orchid plants.     

Disintegration of microorganisms in an industrial horizontal mill of novel design

A continuous high-speed horizontal colloid mill of novel design for use in the microbiological and food industries was tested for the disintegration of cells of Saccharomyces cerevisiae and Candida utilis. The mill consists of a horizontal vessel with round or oval cross sections fitted with a high-speed longitudinal agitator shaft on which are mounted disk agitators, alternating radially and obliquely to the shaft. The mill is partly filled with freely moving grinding elements which, during a continuous operation, are maintained in the vessel by a vibrating annular slot separator. Highly efficient cooling is provided by circulation of cooling fluid through a jacket surrounding the vessel as well as through the agitator shaft and disks. The radial agitator disks impart a radial motion to the grinding elements, while the oblique disks give rise to the axial movement of a substantial part of the elements. The crossing of paths thus achieved gives the mill a very high efficiency. Using a mill of 20 liter nominal capacity, the effects of agitator design, agitator speed, flow rate, and concentration of the cell suspension on the disintegration efficiency and heat production were studied. Ninety per cent of S. cerevisiae cells in a 15% suspension could be broken at a residence time of 2.5 min. The temperature rise did not exceed 8° C. The corresponding figure for C. utilis was 84%. The maximal flow rate was 400 liter/hr. Extrapolation indicates that available industrial mills of 300 liter capacity based on the same design can handle flows of 2000 liter/hr.     

Using CFD simulations and statistical analysis to correlate oxygen mass transfer coefficient to both geometrical parameters and operating conditions in a stirred-tank bioreactor

Optimization of a bioreactor design can be an especially challenging process. For instance, testing different bioreactor vessel geometries and different impeller and sparger types, locations, and dimensions can lead to an exceedingly large number of configurations and necessary experiments. Computational fluid dynamics (CFD), therefore, has been widely used to model multiphase flow in stirred-tank bioreactors to minimize the number of optimization experiments. In this study, a multiphase CFD model with population balance equations are used to model gas–liquid mixing, as well as gas bubble distribution, in a 50 L single-use bioreactor vessel. The vessel is the larger chamber in an early prototype of a multichamber bioreactor for mammalian cell culture. The model results are validated with oxygen mass transfer coefficient (k_La) measurements within the prototype. The validated model is projected to predict the effect of using ring or pipe spargers of different sizes and the effect of varying the impeller diameter on k_La. The simulations show that ring spargers result in a superior k_La compared to pipe spargers, with an optimum sparger-to-impeller diameter ratio of 0.8. In addition, larger impellers are shown to improve k_La. A correlation of k_La is presented as a function of both the reactor geometry (i.e., sparger-to-impeller diameter ratio and impeller-to-vessel diameter ratio) and operating conditions (i.e., Reynolds number and gas flow rate). The resulting correlation can be used to predict k_La in a bioreactor and to optimize its design, geometry, and operating conditions.     

Photosynthetic characteristics of Cymbidium plantlet in vitro

The photosynthetic characteristics of the Cymbidium plantlet in vitro cultured on Hyponex-agar medium with 2% sucrose were determined based on the measurements of CO₂ concentration inside and outside of the culture vessels. The CO₂ measurements were made with a gas chromatograph at a PPF (photosynthetic photon flux) of 35, 102 and 226 mol m^-2 s^-1, a chamber air temperature of 15, 25 and 35°C and a CO₂ concentration outside the vessel of approximately 350, 1100 and 3000 ppm. The net photosynthetic rates were determined on individual plantlets and were expressed on a dry weight basis. The steady-state CO₂ concentration during the photoperiod was lower inside the vessel than outside the vessel at any PPF greater than 35 mol m^-2s^-1 and at any chamber air temperature. The photosynthetic response curves relating the net photosynthetic rate, PPF, and CO₂ concentration in the vessel and chamber air temperature were similar to those for Cymbidium plants grown outside and other C₃ plants grown outside under shade. The results indicate that CO₂ enrichment for the plantlets in vitro at a relatively high PPF would promote photosynthesis and hence the growth of chlorophyllous shoots/plantlets in vitro and that the plantlets in vitro would make photoautotrophic growth under environmental conditions favorable for photosynthesis.Abbreviations C_in CO₂ concentration in the culture vessel - C_out CO₂ concentration outside the vessel (in the culture room) - PPF photosynthetic photon flux     

Automatic evaluation of antifungal volatile compounds on the basis of the dynamic growth process of a single hypha

An automatic analysing system was developed and employed for the evaluation of antifungal activity of volatile compounds in the gas phase. Aspergillus niger was inoculated on agar medium in the reaction vessel. The reaction vessel was incubated at 28° C for 24 h and then a volatile compound was introduced into the vessel either in a batch or flow manner. The antifungal activity of the respective compounds estimated in situ was expressed by the dynamic response parameters of a single hypha. All volatiles tested in the present system inhibited hyphal growth, except linalyl acetate: Limone and geraniol were the most inhibitory. In contrast, linalyl acetate promoted hyphal growth. By definition of the parameters, the fungicidal and fungistatic effects could be distinguished. Correspondence to: H. Matsuoka     

Effect of cycling dissolved oxygen concentrations on product formation in penicillin fermentations

Summary Limitations in mass and momentum transfer coupled with high hydrostatic pressures create significant spatial variations in dissolved gas concentrations in large fermenters. Microorganisms are subjected to fluctuating environmental conditions as they pass through the zones in a stirred vessel or along a closed loop fermenter.A 7-litre fermenter was modified to simulate the dissolved gas and hydrostatic pressure gradients in large vessels.The effect of cycling dissolved oxygen tension (DOT) on penicillin production by Penicillium chrysogenum P1 was investigated. The fermentation was affected by evironmental conditions such as medium composition, pH, size of inoculum, stirrer speed and DOT. Inoculum size below 10% (v/v) and stirrer speeds above 850 rpm caused significant reductions in specific prenicillin production rates (q_pen). q_pen values were measured at different constant DOT levels. Below 30% air saturation q_pen decreased sharply and no production was observed at 10%. Penicillin synthesis was impaired irreversibly below 10% DOT. The same profile was observed at higher stirrer speeds and air flow rates indicating that the effect was a physiological one. Oxygen uptake of the culture was affected significantly below 7% DOT, demonstrating that the critical DOT values for penicillin production and oxygen uptake are two distinct parameters. Carrying out the fermentation at one atmosphere over pressure was found to have no effect. When the dissolved oxygen concentration of the culture medium was cycled around the critical DOT for penicillin production, a considerable decrease in the specific penicillin production rate was observed. The effect was reversible but not transient, indicating a shift in cell metabolism.These results demonstrate the unfavourable effect of fluctuating environmental conditions on culture performance in stirred tanks. They suggest that these effects should be accounted for during strain selection, process development and scale up stages of an industrial process if the productivities in small scale vessels are to be obtained.     

Water and Nutrient Dynamics in Surface Roots and Soils are not Modified by Short-term Flooding of Phreatophytic Plants in a Hyperarid Desert

Little is known of the mechanisms employed by woody plants to acquire key resources such as water and nutrients in hyperarid environments. For phreatophytic plants, deep roots are necessary to access the water table, but given that most nutrients in many desert ecosystems are stored in the upper soil layers, viable shallow roots may be equally necessary for nutrient uptake. We sought to better understand the interaction between water and nutrient uptake from soil horizons differing in the relative abundance of these resources. To this end, we monitored plant water and nutrient status before and after applying flood irrigation to four phreatophytic perennial plant species in the remote hyperarid Taklamakan desert in western China. Sap flow in the roots of five plants of the perennial desert species Alhagi sparsifolia Shap., Karelina caspica (Pall.) Less., Calligonum caput medusea Schrenk, and Eleagnus angustifolia Hill. was monitored using the heat ratio method (HRM). Additionally we measured predawn and midday water potential, foliar nitrate reductase activity (NRA), xylem sap nutrient concentration and the concentration of total solutes in the leaves before, 12 and 96 h after flooding to investigate possible short-term physiological effects on water and nutrient status. Rates of sap flow measured during the day and at night in the absence of transpiration did not change after flooding. Moderately high rates of sap flow (HRM heat pulse velocity, 5–25 cm h⁻¹) detected during the day in soils that had a near zero water content at the surface indicated that all species had contact to groundwater. There was no evidence from sap flow data that plants had utilised flood water to increase maximum rates of transpiration under similar climatic conditions, and there was no evidence of a process to improve the efficiency of water or nutrient uptake, such as hydraulic redistribution (i.e. the passive movement of water from moist soil to very dry soil via roots). Measurements of plant water status, xylem sap nutrient status, foliar NRA and the concentration of osmotically active substances were also unaffected by flood irrigation. Our results clearly show that groundwater acts as the major source of water and nutrients for these plants. The inability of plants to utilise abundant surface soil–water or newly available nutrients following irrigation was attributed to the absence of fine roots in the topsoil layer.     

MAIN ROOT-LATERAL ROOT JUNCTIONS OF TWO DESERT SUCCULENTS: CHANGES IN AXIAL AND RADIAL COMPONENTS OF HYDRAULIC CONDUCTIVITY DURING DRYING

The influence of junctions between main roots and lateral roots on water flow was investigated for the desert succulents Agave deserti and Ferocactus acanthodes during 21 d of drying in soil. Under wet conditions, the junctions did not restrict xylem water flow from lateral roots to main roots, consistent with predictions of axial conductance based on vessel diameters. Embolism caused by drying reduced such axial conductance more at the junctions than in adjoining root regions. Connective tracheary elements at the junctions were abundantly pitted and had large areas of unlignified primary wall, apparently making them more susceptible to embolism than vessels or tracheids elsewhere in the roots. Unlike the decrease in axial conductance, the overall hydraulic conductivity of the junction increased during drying because of an increase in the conductivity of the radial pathway. Despite such increases, main roots may not lose substantial amounts of water to a dry soil during drought, initially because embolism at the junctions can limit xylem flow and later because soil hydraulic conductivity decreases. Moreover, the increased root hydraulic conductivity and a potentially rapid recovery from embolism by connective tracheary elements may favor water uptake near the junctions upon soil rewetting.     

硫化氢在肾脏病中的保护作用

肾脏病发病率逐年增高,已经成为影响人类健康的重要疾病之一。硫化氢是继NO、CO之后的第三种气体信号分子,大剂量有毒害作用,但生理浓度的硫化氢起到舒张血管、抗氧化、抗炎、抗凋亡等重要作用。肾脏病尤其继发性肾脏病如梗阻性肾病、肾移植、糖尿病肾病及高血压性肾损害等与血管病变、氧化应激、炎症密切相关,那么硫化氢与肾脏疾病之间有怎样的关系,本文将就硫化氢在肾脏病中的保护作用做一综述。     

Differential sectoriality in long-distance transport in temperate tree species: evidence from dye flow, <Superscript>15</Superscript>N transport,and vessel element pitting

The capture of patchily distributed nutrients by tree roots has received extensive research, but the fate of those nutrients has not. We performed experiments to determine if nutrient transport within tree species is preferentially transported from specific roots to specific branches. Saplings of five species with contrasting growth requirements were examined: two Betula species (B. papyrifera and B. lenta), Populus tremuloides, and two Acer species (A. saccharum and A. rubrum). To quantify patterns of long-distance transport, we examined the accumulation of safranin-O dye and ¹⁵N in branches when these tracers were applied to isolated lateral roots (dye and ¹⁵N) and to the main root system (¹⁵N). Because transport of nutrients between sectors requires flow through intervessel pit pairs of adjacent xylem vessel elements, we quantified the area of intervessel pits, the number of pits per unit vessel wall area, and the % vessel wall area as pits in Acer and Betula. We found that the two Betula species were integrated (tracers applied to isolated roots were likely to accumulate in all branches), while P. tremuloides and the two Acer species were sectorial (tracer accumulation was more concentrated in particular branches). Betula had the largest number of intervessel pits per unit vessel wall area and the largest percentage of vessel wall area as pits. The high density of bordered pits may explain the ease of tracer movement throughout the two Betula species. Greater integration may allow certain trees (e.g., Betula) to exploit nutritionally patchy environments such as rocky soils, and may alter plant-herbivore interactions.     

Design and performance of a new microcalorimetric system for aerobic cultivation of microorganisms

A twin-type heat-conduction calorimeter for an aerobic fermentation process was designed and constructed fermentor both in batch and continuous runs. The time constant of the calorimeter was 3.3 min. The volumetric oxygen transfer coefficient (k_La) of the vessel was measured by a sulfite oxidation method in a continuous flow system under various rotation speeds and gas flow rates. Sufficient thermal stability of the calorimeter was obtained both in batch and continuous runs within the operation time period.     

Xylem structure and water transport in a twiner,a scrambler,and a shrub of Lonicera (Caprifoliaceae)

Summary Wood structure and function was investigated in different growth forms of temperate honeysuckles (Lonicera spp.). All three species had many narrow vessels and relatively few wide ones, with the measured K _h (flow rate/pressure gradient) approximately 24–55% of the theoretical K _h predicted by Poiseuille's law. Only the twiner, Lonicera japonica, had some vessels greater than 50 m in diameter. The twiner also had the narrowest stem xylem diameters, suggesting the greater maximum vessel diameter hydraulically compensated for narrow stems. Conversely, the free-standing shrub, L. maackii, had the greatest annual increments of xylem but the least percent conductive xylem implying that a great portion of the wood was involved with mechanical support. The scrambler, L, sempervirens had low maximum vessel diameter, high Huber values (= xylem area/leaf area), and low specific conductivities (= measured K _h/xylem area), much like the shrub. The greatest vessel frequency occurred in the scrambler (901 vessels · mm^-2), the highest thus far recorded in vines. The lowest Huber value and highest specific conductivity occurred in the twiner, suggesting little self-support but relatively efficient water conduction. LSC (= measured K _h/leaf area) and maximum vessel diameter of Lonicera vines were near the low end of the range for vines in general.     

Individual and Temporal Variation in Outcrossing Rates and Pollen Flow Patterns in Ceiba pentandra (Malvaceae: Bombacoidea)

Ceiba pentandra is a tropical tree with high rates of selfing in some populations. In mixed‐mating species, variation in selfing is due to changes in adult density or variability of incompatibility systems. The effect of spatial isolation and phenology on selfing rates and pollen flow distances was analyzed using microsatellites in a fragmented population of Ceiba pentandra, in southern Costa Rica. Adult trees within a heterogeneous landscape were classified as grouped or isolated. We compared selfing rates at the individual level, between isolation conditions and 2 yr (2007, 2009), which differed in the number of flowering individuals. Mixed mating was estimated in both years (t_m = 0.624–0.759). Trees mated predominantly by outcrossing, while only a few trees reproduced through selfing. Spatial isolation did not significantly affect outcrossing rates. The progeny of grouped trees was mostly sired by near‐neighbors (<1 km) and by long‐distance pollen flow events in isolated trees. A reduction in the number of flowering individuals in 2009 reduced near‐neighbor matings, increased selfing in grouped trees, and decreased the number of unsampled sires in the progeny. Comparing selfing rates on individuals that flowered in both reproductive periods suggests a flexible mating system. Variation in self‐fertilization rates in this population appears to depend on variation of individual traits, such as genetic variability in self‐incompatibility genes, but it is independent of landscape heterogeneity. In contrast, pollen flow distances depend on local tree density as bats concentrate their foraging between near individuals to maximize energy efficiency.     

Effect of low-concentration chlorine dioxide gas against bacteria and viruses on a glass surface in wet environments

Aims: To evaluate the efficacy of low‐concentration chlorine dioxide (ClO₂) gas against model microbes in the wet state on a glass surface. Methods and Results: We set up a test room (39 m³) and the ClO₂ gas was produced by a ClO₂ gas generator that continuously releases a constant low‐concentration ClO₂ gas. Influenza A virus (Flu‐A), feline calicivirus (FCV), Staphylococcus aureus and Escherichia coli were chosen as the model microbes. The low‐concentration ClO₂ gas (mean 0·05 ppmv, 0·14 mg m^?3) inactivated Flu‐A and E. coli (>5 log₁₀ reductions) and FCV and S. aureus (>2 log₁₀ reductions) in the wet state on glass dishes within 5 h. Conclusions: The treatment of wet environments in the presence of human activity such as kitchens and bathrooms with the low‐concentration ClO₂ gas would be useful for reducing the risk of infection by bacteria and viruses residing on the environmental hard surfaces without adverse effects. Significance and Impact of the Study: This study demonstrates that the low‐concentration ClO₂ gas (mean 0·05 ppmv) inactivates various kinds of microbes such as Gram‐positive and Gram‐negative bacteria, enveloped and nonenveloped viruses in the wet state.     

Effect of gas pressure in the root and stem base zone on methane transport through rice bodies

Ebullition of gas bubbles from the soil surface is, in some cases (e.g., in early growth stage of rice), one of the major pathways for methane transport from rice paddies to the atmosphere. However, the role of the gas phase (entrapped gas) in the paddy soil in plant-mediated methane transport, which is the major pathway for methane emission, has not been clarified. To clarify the effect of the gas phase below ground on the methane emission rate through rice plants, we partly exposed the root and stem base of hydroponically grown rice to a high concentration of methane gas at various gas pressures, and immersed the rest of the roots in a solution with a high methane concentration. The methane emission rate was measured from the top of the rice plant using a flow-through chamber method. The methane emission rate drastically increased with a small increase in gas pressure in the gas phase at the root and stem base zone, with about a 3 times larger emission rate being observed with 10 × 10^-3 atm of extra pressure (corresponding to 10 cm of standing water in rice paddy) compared to no extra pressure. However, when alginate was applied to the stem near the base to prevent contact with the gas phase, the methane emission rate did not increase with increasing gas pressure. On the other hand, from observations in the rice paddy, it was found that the gas is entrapped near the surface (e.g., at a depth of 1 cm) and the gas entrapped in the soil would come into direct contact with a part of the stem near the base of the rice plant. Thus, the gas entrapped in the soil could enter into the rice body directly from the part of the stem near the base which is beneath the soil surface due to gas pressure in the gas phase resulting from the pressure exerted by the standing water. Hence, this mechanism involving the entrapped gas could play an important role in methane emission from rice paddy by affecting the plant-mediated methane transport as well as ebullition of gas bubbles.     

Miniaturized systems for evaluating enzyme activity in polymeric membrane bioreactors

Enzyme‐coated polymeric membranes are versatile catalysts for biofuel production and other chemical production from feedstock, like plant biomass. Such bioreactors are more energy efficient than high temperature methods because enzymes catalyze chemical reactions near room temperature. A major challenge in processing plant biomass is the presence of lignin, a complex aromatic polymer that resists chemical breakdown. Therefore, membranes coated with enzymes such as laccase that can degrade lignin are sought for energy extraction systems. We present an experimental study on optimizing an enzyme‐based membrane bioreactor and investigate the tradeoff between high flow rate and short dwell time in the active region. In this work, zero flow rate voltammetry experiments confirm the electrochemical activity of Trametes versicolor laccase on conductive polymer electrodes, and a flow‐through spectroscopy device with laccase‐coated porous nylon membranes is used with a colorimetric laccase activity indicator to measure the catalysis rate and percent conversion as a function of reactant flow rate. Membrane porosity before and after laccase coating is verified with electron microscopy.